1. Field of the Invention
The present invention relates generally to the fields of molecular genetics, cancer biology, cancer prevention, and cancer chemotherapy. More specifically, the present invention relates to the c-DNA cloning and nucleotide sequencing of a novel tocopherol associated protein, referred to as TAP-38, from human normal and breast cancer cells, and the use of molecular/immune technologies to demonstrate that this protein is relevant to the ability of novel tocopherol compounds to induce cancer cells to undergo growth arrest via inhibition of DNA synthesis, induction of cellular differentiation, and induction of apoptosis.
2. Description of the Related Art
The regulatory controls of pro-life (cell proliferation) and pro-death (apoptosis) are extremely complex and involve multiple intracellular signaling pathways and multiple interacting gene products. Cancer cells may exhibit multiple defects in normal regulatory controls of cell proliferation, such as enhanced expression of genes, which allow them to increase in number. In addition to enhanced expression of pro-life genes, cancer cells down-regulate genes and their products that control pro-death signals, resulting in the accumulation and potential metastasis of life threatening cancer cells. Thus, combinations of unregulated cell proliferation and suppression of death inducing signaling pathways give cancer cells both growth and survival advantages.
Whether a cell increases in numbers or not depends on a balance of expression of negatively-acting and positively-acting growth regulatory gene products, and the presence or absence of functional cell death signaling pathways. Negative-acting growth regulatory genes contribute to blockage of cells in the cell cycle. Positive-acting growth regulatory genes stimulate cells to progress through the cell cycle. Genes involved in apoptosis can be either proapoptotic or antiapoptotic, and the dynamic balance between them determines whether a cell lives or dies. Cancer cells, in order to survive and increase their numbers, undergo a series of mutational events over time that remove regulatory controls that give them the ability to grow unchecked and survive even in the presence of proapoptotic signals, and develop attributes that permit them to escape detection and removal by the immune response defense system. Cancers may cause death of individuals unless removed by surgery or effectively treated with drugs.
A wide variety of pathological cell proliferative conditions exist for which novel therapeutic strategies and agents are needed to provide therapeutic benefits. These pathological conditions may occur in almost all cell types capable of abnormal cell proliferation or abnormal responsiveness to cell death signals. Among the cell types that exhibit pathological or abnormal growth and death characteristics are (1) fibroblasts, (2) vascular endothelial cells, and (3) epithelial cells. Thus, novel methods are needed to treat local or disseminated pathological conditions in all or almost all organ and tissue systems of individuals.
Most cancers, whether they are male specific, such as prostate or testicular, or female specific, such as breast, ovarian or cervical, or, whether they affect males and females equally, such as liver, skin or lung, with time undergo increased genetic lesions and epigenetic events, and eventually become highly metastatic and difficult to treat. Surgical removal of localized cancers has proven effective only when the cancer has not spread beyond the primary lesion. Once the cancer has spread to other tissues and organs, the surgical procedures must be supplemented with other more specific procedures to eradicate the diseased or malignant cells. Most of the commonly utilized supplementary procedures for treating diseased or malignant cells such as chemotherapy or bioradiation are not localized to the tumor cells and, although they have a proportionally greater destructive effect on malignant cells, often affect normal cells to some extent.
Some natural vitamin E compounds, and some derivatives of vitamin E have been used as proapoptotic and DNA synthesis inhibiting agents. Structurally, vitamin E is composed of a chromanol head and an alkyl side chain. There are eight major naturally occurring forms of vitamin E: alpha (α), beta (β), gamma (γ), and delta (δ) tocopherols and α, β, γ, and δ tocotrienols. Tocopherols differ from tocotrienols in that they have a saturated phytyl side chain rather than an unsaturated isoprenyl side chain. The four forms of tocopherols and tocotrienols differ in the number of methyl groups on the chromanol head (α has three, β and γ have two and δ has one).
RRR-α-tocopheryl succinate is a derivative of RRR-α-tocopherol that has been structurally modified via an ester linkage to contain a succinyl moiety instead of a hydroxyl moiety at the 6-position of the chroman head. This ester linked succinate moiety of RRR-α-tocopherol has been the most potent form of vitamin E affecting the biological actions of triggering apoptosis and inhibiting DNA synthesis. This form of vitamin E induces tumor cells to undergo apoptosis, while having no apoptotic inducing effects on normal cells. The succinated form of vitamin E is effective as an anticancer agent as an intact agent; however, cellular and tissue esterases that can cleave the succinate moiety, thereby converting the succinate form of RRR-α-tocopherol to the free RRR-α-tocopherol, render this compound ineffective as an anticancer agent. RRR-α-tocopherol exhibits neither antiproliferative nor proapoptotic biological activity in cells of epithelial or immune origin. Attachment of the succinate moiety to the C-6 carbon on the chromonal ring of RRR-α-tocopherol via an ether linkage provides stable tocopherol based apoptotic inducing compounds that can not be rendered ineffective since cells do not have etherases to clip off the succinate moiety.
To understand, in part, the mechanisms of action of tocopherols and tocotrienols as anticancer agents requires an understanding of their binding and their inter- and intra-cellular transport, via proteins that specifically interact with these compounds. It is well established that very low density lipoproteins (VLDLs) are loaded with RRR-α-tocopherol in the liver (2) allowing for the entrance of RRR-α-tocopherol into circulation. The liver protein alpha-tocopherol transport protein(-α-TTP) has been shown to be involved in this process (3, 4). The sequence of α-TTP has been reported and the protein exhibits specificity for the RRR-α-tocopherol form, compared to the other isomers and forms of vitamin E (5–7). Another small molecular weight protein has been reported to be present in various tissues (8–10) however, the sequence or the role of this protein remains unidentified.
Recently, a protein was identified from humans and from bovine as having specificity for the RRR-forms of tocopherol (11, 12). The protein is 46 KDa in mass and has a characteristic CRAL-TRIO domain, a domain involved in binding to hydrophobic ligands (13). This protein was called tocopherol-associated protein (TAP-46). A more recent paper, however, identified the identical protein as having a role in enhancing cholesterol biosynthesis by promoting the conversion of squalene to lanosterol and called the protein supernatant protein factor (SPF) (14).
The prior art is an effective means of inhibiting undesirable or uncontrollable cell proliferation in a wide variety of pathophysiological conditions while having no to little effect on normal cells. The present invention fulfills this long-standing need and desire in the art.